During FY14 we accomplished the following: 1. We completed additional ITRAK studies with CD4+ T cells from 16 individuals. We found that several mitochondrial proteins were expressed at higher levels in cells from older individuals. Further validation and mitochondrial quantitation is in progress. 2. We carried out studies to investigate the mechanistic basis for elevated NF-&#954;B gene expression signature in CD4+ T cells. We found that a subset of NF-&#954;B target genes were up-regulated in a PI3K-dependent manner, which did not involve mitochondrial reactive oxygen species. These observations provide strong evidence that elevated PI3K activity in CD4+ T cells from older individuals contributes to age-associated chronic inflammation. 3. We established a protocol to separate 10 blood cell types from peripheral blood mononuclear cells for transcription, proteomic and epigenetic analyses. This involved initial separation of CD4+, CD8+, B cells, monocytes and natural killer cells by magnetic beads, followed by flow cytometry. Aliquots of sorted cells were frozen for RNA, proteins and genomic DNA preparation. We have tested and found that good quality RNA and proteins can be prepared from these frozen aliquots.